Abstract

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the μ-opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the δ-agonist deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for the μ- and δ-opioid receptor agonists in both functional assays. The highest potency at the μ-receptor was observed for endomorphin-1, endomorphin-2, and [d-1-Nal3]morphiceptin, whereas deltorphin-II was the most potent δ-receptor agonist. In the aequorin assay, the μ- and δ-agonist-triggered luminescence was inhibited by the opioid antagonists naloxone and naltrindole, respectively. We can conclude that the use of the aequorin assay for new μ- and δ-receptor-selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in the screening of opioid analogs.

Current methods for the identification and characterization of new ligands at the three main opioid receptor types (μ, δ, κ) typically use binding assays, where a radioligand with high affinity to one receptor type is displaced by a tested analog. Apart from binding studies, different functional assays can also be used to determine relative potency and efficacy of new analogs at the opioid receptors. The most popular functional assay used for opioid activity determination in vitro is performed on isolated tissue preparations. This assay is based on inhibition of electrically evoked contractions of guinea pig ileum (GPI) and mouse vas deferens (MVD). The opioid effect in the GPI preparations is mainly mediated by the μ-receptors, whereas the predominant receptors of the MVD are of the δ-type. The GPI/MVD assay makes it possible to determine the μ- and δ-receptor interactions, which is the main focus in standard tests for opioid activity.

In the present study, we used an alternative functional assay, which does not involve radioactivity or animal tissues and allows high-throughput screening of opioid peptide analogs. This assay is based on the recombinant mammalian cell line expressing an opioid receptor together with a luminescent reporter protein.

Opioid receptors belong to a large family of the G protein-coupled receptors (GPCR), which on stimulation by a ligand induce the activation of the phospholipase C, with the subsequent generation of the primary second messenger metabolites: diacylglycerol and inositol 1,4,5-trisphosphate, followed by a release of calcium from intracellular stores. These agonist-induced receptor-mediated changes in calcium levels can be quantitatively assessed and used to determine agonist potency and efficacy.

A number of groups (Stables et al., 1997; Ungrin et al., 1999; George et al., 2000; Torfs et al., 2002c; Niedernberg et al., 2003) have reported the use of the photoprotein aequorin (Shimomura et al., 1962) as a sensitive detector of increases in Ca2+ levels. Apoaequorin is a 21-kDa photoprotein, isolated from the jellyfish Aequorea victoria, which forms a bioluminescent complex when linked to the chromophore cofactor coelenterazine (Shimomura, 1995). The binding of Ca2+ to this complex results in an oxidation reaction of coelenterazine, followed by the production of apoaequorin, coelenteramide, CO2, and light with a λmax of 469 nm, which can be detected by conventional luminometry (Stables et al., 1997). Since its discovery in the 1960s, aequorin has been used as an intracellular calcium indicator. The first applications of aequorin involved its microinjection into cells. The cloning of its gene in 1985 opened the way to the stable expression of apoaequorin in different cell lines (Inouye et al., 1985). In a cell line expressing recombinant apoaequorin, reconstitution of aequorin can be simply obtained by the addition of coelenterazine to the medium (Brini et al., 1995).

The aim of the present study was to characterize the relative agonist potency and efficacy of three structurally related μ-selective opioid peptides, endomorphin-1, endomorphin-2, and morphiceptin and their position 3-substituted analogs, as well as a δ-agonist, deltorphin-II, in the aequorin assay performed on the genetically engineered Chinese hamster ovary (CHO) cell lines CHO-MOR-Aeq and CHO-DOR-Aeq coexpressing, respectively, μ- or δ-opioid receptors and apoaequorin. A comparison with the data obtained previously from conventional opioid receptor binding and functional GPI/MVD assays was also performed.

Materials and Methods

Peptide Synthesis. Peptides were synthesized by standard solidphase procedures as described before (Fichna et al., 2004) using techniques for N-(9-fluorenyl)methoxycarbonyl-protected amino acids on 4-methylbenzhydrylamine Rink peptide resin (100–200 mesh, 0.8 mM/g; Novabiochem, San Diego, CA). Twenty percent piperidine in dimethylformamide was used for deprotection of N-(9-fluorenyl)-methoxycarbonyl groups, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-methyluronium tetrafluoroborate was used to facilitate coupling. Simultaneous deprotection and cleavage from the resin were accomplished by treatment with trifluoroacetic acid (TFA)/triisopropylsilane/water (95:2.5:2.5) for 3 h at room temperature. Crude peptides were purified by reverse phase high-performance liquid chromatography on a Vydac C18 column (1 × 25 cm) using the solvent system of 0.1% TFA in water (A)/80% acetonitrile in water containing 0.1% TFA (B) and a linear gradient. Calculated values for protonated molecular ions were in agreement with those obtained using fast atom bombardment mass spectrometry.

Aequorin Charging Protocol. The CHO cells were grown until 90% confluent monolayers were obtained. Cells in mid-log phase were detached from culture flasks by flushing with phosphate-buffered saline/EDTA (5 mM EDTA), centrifuged (5 min, 150g), and resuspended in “bovine serum albumin (BSA) medium” (Dulbecco's modified Eagle's medium/F-12 with HEPES and 0.1% BSA, without phenol red) at a concentration of 5 × 106 cells/ml. Cells were incubated at room temperature for 4 h in BSA medium supplemented with 5 μM Coelenterazine h (Molecular Probes, Leiden, The Netherlands). After coelenterazine loading, the cells were diluted 10 times in the same medium and incubated for 1 h.

Aequorin Assay. Test substances were dissolved in 50 μl of BSA medium and pipetted into the wells of the white 96-well plates. Light emission was recorded (EG&G Microplate Luminometer LB96V, Berthold, Germany) for 30 s immediately after injection of 50 μl of cell suspension (i.e., 25,000 cells) into each well. Cells were then lysed by a second injection of 50 μl of 0.3% Triton X-100, followed by a 15-s monitoring period. Luminescence data (peak integration) were calculated using Winglow software (PerkinElmer, Boston, MA), which was linked to the Microsoft Excel (Redmond, WA) program. Results are expressed as the fractional luminescence, i.e., the ratio of the agonist-generated signal and the total luminescence (agonist + lysed cells), thereby correcting for potential well-to-well variation in the number of injected cells (Knight et al., 2003).

Data Analysis. Statistical and curve-fitting analyses were performed using Prism 4.0 (GraphPad Software Inc., San Diego, CA). Three independent experiments for each assay were carried out in duplicate. All the values are expressed as mean ± S.E.M.

Results

Calcium Measurements and Concentration-Response Curves. Recombinant mammalian cell lines, CHO-MOR-Aeq and CHO-DOR-Aeq, expressing, respectively, the μ- or δ-opioid receptors and apoaequorin were used to study agonist-induced bioluminescent responses. Three structurally related μ-selective opioid peptides, endomorphin-1, endomorphin-2, and morphiceptin and their position 3-substituted analogs, as well as a δ-agonist, deltorphin-II, were chosen for the study. The concentration-response curves for the Ca2+ responses in the μ-receptor-expressing cells are depicted in Fig. 1. Calculated EC50 values and maximal Ca2+ increases are listed in Table 1. A maximal increase in Ca2+ concentration at the μ-opioid receptor was obtained with endomorphin-1, endomorphin-2, morphiceptin, and analogs 4 and 5. For analogs 6 through 8, the maximal Ca2+ stimulation reached only approximately 75% of the level found with peptides 1 through 5, indicating that these analogs behaved as partial agonists at the μ-opioid receptor. Despite the lower maximal responses, activity of analogs 6 through 8 was detectable at concentrations as low as 10–11 to 10–12 M. Concentration-response curves for the Ca2+ response induced in the δ-opioid receptor-expressing cells are shown in Fig. 2. Deltorphin-II behaved as a potent δ-agonist, with the EC50 value of 0.0017 ± 0.0001 nM and a maximal Ca2+ stimulation of 86% (100% = total luminescence as determined after cell lysis). All of the other tested peptides behaved as partial agonists, reaching 51 to 75% of the total luminescence level. Their EC50 values were at least two or three orders of magnitude higher than the value obtained for deltorphin-II.

Concentration-response curves for the calcium increase induced by μ- and δ-selective opioid peptides in the CHO-MOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

Concentration-response curves for the calcium increase induced by μ- and δ-selective opioid peptides in the CHO-DOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

Effect of Opioid Antagonists on Agonist-Evoked Responses in CHO-MOR-Aeq and CHO-DOR-Aeq Cells. The opioid antagonists naloxone and naltrindole were used to assess their influence on the agonist-induced bioluminescent responses in the μ- and δ-opioid receptor-expressing cells, respectively. Naloxone produced a concentration-dependent rightward shift of concentration-response curves for the μ-selective peptides in CHO-MOR-Aeq cells. The exemplary results for endomorphin-1 and the most potent μ-agonist, analog 5, are shown in Fig. 3. The effect produced by deltorphin-II was blocked by pretreatment with the δ-antagonist naltrindole (Fig. 4).

Concentration-dependent effect of naloxone on the concentration-response curves for the calcium increase induced by endomorphin-1 1 (A) and [d-1-Nal3]morphiceptin 5 in the CHO-MOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

Here we have selected endomorphins and morphiceptin, as well as their position 3-modified analogs, to study their relative potency and efficacy at the μ- and δ-opioid receptors expressed in CHO cells. For comparison, a highly δ-selective peptide, deltorphin-II (Tyr-d-Ala-Phe-Glu-Val-Val-Gly-NH2), originating from amphibian skin (Kreil et al., 1989), has also been studied. We have used a functional assay based on agonist-evoked changes of intracellular Ca2+ levels in CHO cells stably transfected with the opioid receptor (μ or δ) and apoaequorin cDNA. The light-emission responses obtained in the aequorin-based assay for all of the tested peptides were concentration-dependent, and they were inhibited by opioid antagonists.

Concentration-dependent effect of naltrindole on the concentration-response curves for the calcium increase induced by deltorphin-II 9 (A) and [d-ClPhe3]morphiceptin 7 in the CHO-DOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

The results from this study were compared with our previous data obtained in conventional functional assay (GPI/MVD) and with a traditional binding assay on rat brain membrane preparations (Table 2). In both functional assays, the most potent compounds at the μ-receptor were endogenous ligands, endomorphin-1, endomorphin-2, and morphiceptin analog 5 (Tyr-Pro-d-1-Nal-Pro-NH2). These three peptides were almost equipotent in the GPI/MVD assay but showed a significant difference in potency in the calcium assay. Endomorphin-1 was the most potent and selective peptide of the series. Analog 5 was 7 times less potent, whereas the second endogenous ligand, endomorphin-2, was 180 times less potent than endomorphin-1. Morphiceptin was the only peptide showing much higher selectivity in the GPI/MVD assay than in the calcium assay. Analog 7 (Tyr-Pro-d-ClPhe-Pro-NH2) was the only analog with higher potency for the δ-receptor over the μ-receptor in both tests.

Although the rank order of potencies of tested peptides was comparable in both functional assays, the EC50 values observed in the bioluminescent calcium assay were several orders of magnitude lower than the IC50 values observed in the GPI/MVD assay and in the radioligand binding assay on rat brain membrane preparations. This clearly illustrates the extraordinary sensitivity of the cell-based aequorin assay, which therefore can differentiate the potency of opioid analogs much better than the GPI/MVD assay. This may be because cell clones expressing a specific receptor type ensure much higher selectivity of this assay system than the traditional bioassays on complex tissue preparations.

In conclusion, the selected group of peptides, including well known ligands of high μ-affinity (endomorphin-1 and endomorphin-2), moderate μ-affinity (morphiceptin), high δ-affinity (deltorphin-II), and position 3-substituted analogs of endomorphins and morphiceptin, which in the binding experiments exhibited different degree of μ- or δ-affinity, were tested on the cell lines expressing μ- or δ-opioid receptor and a reporter protein, apoaequorin. The obtained results allowed us to differentiate all of the tested peptides in terms of their potency to a much greater degree than was possible using a functional assay on tissue preparations.

Acknowledgments

We thank Dr. M. Detheux (Euroscreen) for advocating the use of the cell lines and Jozef Cieslak and Sofie Van Soest for excellent technical assistance.

Footnotes

This work was supported by a grant for Bilateral Scientific Cooperation between Flanders and Poland (BIL03/18). In addition, the authors gratefully acknowledge the Belgian Interuniversity Attraction Poles Programme (IUAP/PAI P5/30, Belgian Science Policy) and the Fonds voor Wetenschappelijk Onderzoek—Vlaanderen (FWO) for financial support. J. Poels is supported by the FWO as a postdoctoral research associate.